For single-molecule measurements, Cascade was labeled with a biotin on the N terminus of its Cse1 subunit and immobilized to the surface of a microscope slide via a biotin-streptavidin linkage (Figure 1A). Dye-labeled dsDNA targets were added to the slide, and individual binding events were imaged in real time with a total internal reflection fluorescence (TIRF) microscope (Figure 1A). DNA constructs consisted of a protospacer, a PAM, and an additional 15 base pair flank (Figure 1B). The target strand (complementary to the crRNA) was labeled with an acceptor dye (Cy7) at protospacer position +9, whereas the nontarget strand was labeled with a donor dye (Cy3) at protospacer position +17. These labeling positions yielded a FRET value of ~0.65 (named
2
Figure 1. Two Binding Modes of Cascade Revealed by a Single-Molecule FRET Assay. (A) Schematic of a single- molecule FRET experiment used to monitor binding of Cascade to target DNA substrates. (B) The bona fide target construct consists of a 15 bp flank (black), a PAM(orange), and a protospacer (green), with its seed highlighted in blue. Cy7 (red star) was attached to position +9 of the target strand and Cy3 (green star) to position +17 of the nontarget strand. (C) A representative time trace of donor (Cy3, green) and acceptor (Cy7, red) fluorescence and corresponding FRET (blue) exhibiting the long-lived binding of the bona fide target. High FRET (~0.84, named EI for FRET efficiency of an intermediate state) exhibited upon binding is followed by low
FRET (~0.44, named EO for FRET efficiency of an open state). DNA was added at time = 10 s. (D) A representative
time trace exhibiting the short-lived binding of the bona fide target exhibits two FRET states (EO~0.44 and
EC~0.65; EC is for FRET efficiency of a closed state). The duration of each state is measured as the dwell time
(Δτ). DNA was added at time = 10 s. (E) The FRET distribution of the bona fide target DNA alone (light blue) or after equilibration with immobilized Cascade (purple) with peaks at EC (0.65) and EO (0.44), respectively (derived
from Gaussian fit, black line). Data were obtained from five fields of view each. (F) A histogram of the initial FRET upon binding (average of first 1.5 s of each event) of the bona fide target exhibits three peaks at FRET = EO (0.44), EC (0.65), EI (0.84) (derived from Gaussian fit, black line). (G) The survival rate of events that start at
EI (0.84) was fitted using a single (light blue color) and a double (black color) exponential curve. The double
exponential fit resulted in two characteristic times (25.9 and 1040 s). (H) The dwell time distribution of EI (0.84)
state of bona fide target binding with mean Δτ0.84 (derived from single exponential fit, black line). Error
represents standard deviation (three individual data sets).
Recent in vivo studies have revealed an additional functionality of CRISPR-Cas immunity. When facing ‘‘escape mutants,’’ previously targeted sequences that bear mutations in their PAM and/or protospacer, Cascade initiates a response called priming wherein the CRISPR-Cas system acquires new spacer sequences from the mutant at an elevated rate to restore immunity (Datsenko et al., 2012; Fineran et al., 2014; Li et al., 2014; Richter et al., 2014). High-throughput plasmid loss assays of a randomized PAM and protospacer library have revealed that priming is a robust process, tolerating up to 13 mutations in the PAM and protospacer sequence (Fineran et al., 2014). Even though Cascade is essential for priming, its role in this process is poorly understood. Intriguingly, biochemical studies have shown that a single-point mutation in the PAM or seed sequence leads to a drastic decrease in the binding affinity of Cascade (Semenova et al., 2011). Therefore, it is puzzling how Cascade is able to associate with these mutated substrates despite its low affinity and, further, how it distinguishes these mutated substrates from bona fide targets to initiate priming.
Single-molecule fluorescence is a powerful tool for elucidating the intricate mechanistic details of complex protein-nucleic acid interactions (Ha, 2014; Joo et al., 2013; Juette et al., 2014; Robinson and van Oijen, 2013; Schuler and Hofmann, 2013). To dissect Cascade’s two distinct functional roles, we developed a single-molecule FRET assay to monitor the interaction of Cascade with bona fide and mutated substrates. Real-time observation of Cascade-target interactions revealed that an initial recognition complex proceeds to a stable R-loop only if the crRNA makes an extensive match with the target. In addition to this ‘‘canonical binding mode,’’ we identified an alternative binding mode of Cascade that is triggered by partial complementarity to a target. Using an in vivo assay, we validated that this binding mode enables Cascade to probe mutated DNA substrates and consequently initiate priming.
Results
Single-Molecule Observation of Cascade Target Binding
For single-molecule measurements, Cascade was labeled with a biotin on the N terminus of its Cse1 subunit and immobilized to the surface of a microscope slide via a biotin-streptavidin linkage (Figure 1A). Dye-labeled dsDNA targets were added to the slide, and individual binding events were imaged in real time with a total internal reflection fluorescence (TIRF) microscope (Figure 1A). DNA constructs consisted of a protospacer, a PAM, and an additional 15 base pair flank (Figure 1B). The target strand (complementary to the crRNA) was labeled with an acceptor dye (Cy7) at protospacer position +9, whereas the nontarget strand was labeled with a donor dye (Cy3) at protospacer position +17. These labeling positions yielded a FRET value of ~0.65 (named
EC for a FRET state which represents a closed conformation of dsDNA between nt 9 and 17) (Figure 1E) as
measured by immobilization of the DNA alone (see Experimental Procedures and Table S1). Control experiments showed that dye labeling of the DNA at protospacer positions +9 and +17 did not appreciably affect the target binding reaction of Cascade (Figure S1F). We first explored Cascade’s interaction with a bona fide target DNA, a substrate that triggers interference in vivo. This substrate contains a protospacer with perfect complementarity to the crRNA and an interference-permissive PAM (named ‘‘interfering PAM’’) (Figure 1B) (Fineran et al., 2014; Westra et al., 2012a). After equilibration of the DNA with the immobilized Cascade, the measured FRET distribution exhibited one major peak centered at 0.44 (named EO for a FRET state which
represents an open conformation of dsDNA between nucleotides 9 and 17), a decrease from the starting value of EC (0.65) (Figure 1E). This decrease in FRET is consistent with the expected open DNA conformation resulting
from R-loop formation upon Cascade binding. A similar decrease in FRET was observed upon exchanging the position of the donor and acceptor dyes (Figure S1C) or when Cascade was prebound to the DNA prior to immobilization (Figure S1D), indicating that the observed decrease in FRET was not due to a protein- or surface- induced photophysical effect.